- Information
- AI Chat
Protoplast Culture and Somatic Hybridization
Jamia Millia Islamia
Related documents
- British Orientalism-History Assignment
- Politics, Political Science, Political Thought, Theory An Orientation
- A Brife Note On Nationalism's advantages and disadvantages
- Emergence and decline of arab nationalism
- Exploration of Rabindranath Tagore's The Home and the World as Socio-political Novel
- Muslim Mystics AND SUFI Silsilahs
Preview text
Cut
Protoplast Culture and Somatic Hybridization
In plants, where fairly distant species could be crossed, it has always not been possible to obtain full hybrids between desired individuals because of sexual incompatibility barriers. This has often proved to be a serious handicap in crop improvement programs through hybridization. With the ability of isolating protoplasts from plant cells using enzymes in the early 1960 by E. C. Cocking, the interest in genetic modification of somatic cells in higher plants has developed. Within a decade, Takebe et al. (1971) regenerated completes plants from leaf protoplasts of tabacco increased the potential of prtoplast culture techniques.
Not only isolated protoplasts but their fusion product, a somatic hybrid, can also be regenerated into whole plants. Plant protoplasts can also take up foreign DNA, through their naked plasma membrane, under specific chemical and physical treatments. Protoplasts also provide an experimental system for a wide range of biochemical and molecular studies ranging from investigations into the growth properties of individual cells to membrane transport.
Present chapter describes the methods of protoplast isolation, purification, culture techniques as also protoplast fusion techniques and somatic hybrid selection procedures.
I. PROTOPLAST ISOLATION
The term protoplast refers to the spherical plasmolysed content of plant cell enclosed by plasma membranes or naked cell without cell wall. Before going into the details about
protoplast culture, it is important to isolate as gently and as quickly as possible viable and uninjured protoplasts. The two methods for isolation of protoplasts are: mechanical and enzymatic method.
Mechanical Isolation Klercker (1892) was the first to isolate protoplasts from plasmolysed cell of Stratiates aloides. These studies were later extended for protoplasts isolation from tissues of onion bulbs. Scales were immersed in 1 M sucrose until the protoplast shrunk away from their enclosing wall and then the plasmolysed tissue was cut with a sharp knife at such a thickness that only the cell walls are cut without damaging the protoplasts in strips. The protoplasts were released by osmotic swelling when these strips of the tissue are placed in low concentration sucrose solution (Fig. 1).
A B
Fig. 1. When tissue is cut at the dotted lines (A) with a sharp razor blade, some cells release uncut complete protoplast and rest of the cells produced broken dead protoplasts as shown in Figure 1B marked with stars (*).
I Enzymatic Isolation This method involves the use of enzymes to dissolve the cell wall for releasing protoplasts. Initial studies were carried out on protoplasts isolation of yeast cell by digesting the cell wall using gastric juices obtained from the snail, Halix poneatia. However, credit of developing high yield protoplast isolation technique from higher plant protoplasts goes to Cocking (1960). He employed a crude cellulase preparation from the fungus Myrothecium verrucaria to dissolve cell wall and release the protoplasts from tomato roots. Later this method with suitable modifications and using purified enzymes has been extensively used by other group of workers. The enzymatic method could be used as a one step method (direct method), or as a two-step method (sequential method).
In the one step method, protoplasts are isolated directly from the tissue by using two enzymes, cellulase and pectinase, simultaneously. While, in the two-step method, cells are first isolated from callus or tissue by using pectinase and to this cell suspension cellulase is added to digest the cell wall and release protoplasts.
An enzymatic solution used for protoplast isolation contains the enzymes and a sugar as an osmoticum to prevent the plasma membrane from rupturing. Some salts and nutrients are also used as osmoticum. Generally 50 mM CaCl 2 is added to increase the stability of released protoplasts (Rose, 1980). The enzyme solution is filter sterilized through a 0μ filter membrane and sterilized tissue cut into small pieces is added. Incubation for some time releases protoplasts from the tissue.
PROTOPLAST PURIFICATION
The successful culture of protoplasts requires a pure population of intact and viable protoplasts at a high yield. So the protoplasts require to be purified by removing the undigested material (debris), burst protoplasts and enzymes.
Removal of Debris and Enzymes Debris can be removed from the protoplast suspension by filtering the preparation through a steel or nylone mesh of 100μ pore size. Enzyme is removed by centrifuging the protoplast suspension at 600 rpm for 5 minutes. The protoplasts settle to the bottom of the centrifuge tube while the supernatant is removed with the help of a pipette. The protoplasts are then re- suspended in a washing medium containing an osmoticum only or osmoticum with nutrient medium or hydrated calcium chloride. The suspension is centrifuged again to settle the protoplasts and the washing medium is decanted. Traces of enzyme are removed by washing the protoplasts twice or thrice with the medium.
Removal of Broken Protoplasts Intact protoplasts are separated from the broken debries by suspending the protoplast preparation in 20-40% sucrose solution and centrifugation at 350 rpm for three minutes. Intact protoplasts collect at the top of the sucrose solution and are carefully removed with a pipette (Gregory and Cocking, 1965; Power and Cocking, 1970; Evans et al. , 1972).
in presence of Ca++ or Mg++ ions showed a greater capacity for cell wall regeneration as compared to protoplasts isolated in the absence of these ions (Rose, 1980).
PROTOPLAST CULTURE
After viability test the protoplasts are cultured at a known density. Different methods have been used for culturing the isolated protoplasts.
Suspension Culture
In this method protoplasts are suspended in a liquid medium with a suitable concentration of osmoticum. Protoplasts at a density of 10 5 /ml is generally plated on 25-50 ml of medium in an Erlenmeyer flask. The cultures are shaken slowly to provide sufficient aeration for growth. Sometimes shaking can cause bursting of protoplasts, so rpm of shaking required for a given species should be standardized. Evans and Cocking (1975) suggested that 2 ml suspension of protoplasts could be cultured in 25 ml flasks to facilitate aeration. Vasil (1976) suggested the addition of ficoll to the medium to keep protoplasts floating and thereby allowing better growth.
Hanging Drop Method
Kao and his group developed the hanging drop method in 1970 which was subsequently used by others (Bawa and Torrey, 1971). In this method, a suspension of protoplasts at a density of 104 /ml to 10 5 /ml is placed as 50 μl drops in plastic Petri plates, sealed with parafilm and incubated in an inverted position at 25-30°C under lw light intensity (100-500 lux) or even in dark. After cell wall regeneration and the initiation of cell division, fresh medium is added to make cell suspension. The small size of drop helps in providing enough aeration to protoplasts (Vasil, 1976).
Agar Plating Method
The method is almost similar to the one developed by Bergmann (1960) to grow callus cells of tobacco and beans and was first employed for the protoplast culture by Nagata and Takabe (1971). The major advantages of this technique are: (i) a large number of protocols can be handled simultaneously, and (ii) plating efficiency can be determined easily. At the same time the major drawback of this technique is that after regeneration of cell wall and induction of cell division, osmotic potential of the medium can not be altered by addition of fresh medium lacking osmotic stabilizer, since the initial medium is semi-solid. However, this problem can be solved by transferring blocks of agar (in which protoplasts are cultured initially) to a fresh medium with lesser concentration of osmoticum or altogether in its absence. This technique has been modified in different ways viz., as feeder technique to support division of cells plated at low density (Raveh et al., 1973), as micro vessel (Button, 1978), and as multiple drop arrays (Potrykus et al., 1979).
Micro Culture Technique
This technique developed by Jones et al. (1960) for culturing isolated cells was used by Vasil and Hilderbrandt (1965) to raise tobacco plants from isolated cells. Micro culture technique has been successfully used for culturing protoplasts of tobacco and Petunia (Durand et al., 1973). A drop of culture medium containing one or more protoplasts is put on a microscopic slide. On either sides of this microscopic slide are kept two cover slips. A third one is put over these two cover slips to shield the protoplasts suspensions.
Multidrop Array (MDA) Technique
This is a further improvement of hanging drop method and allows screening of large number of hormonal and nutritional factors (up to 4900) using small amount of plant material (Potrykus et al., 1979)
FACTORS AFFECTING PROTOPLASTS CULTURE
Successful growth and regeneration of protoplasts is dependent on a number of factors ranging from the status of the donor plant to the culture conditions.
cytoplasm of two cells is the plasma membrane. After lot of refinements the techniques for protoplast fusion became important to produce hybrids from sexually incompatible species. Protoplast fusion is possible even between a plant cell and an animal cell and thus can have wider application. The process of somatic hybridization involves: (i) protoplast isolation, (ii)protoplast fusion, (iii) selection of somatic hybrids, and (iv) culture of somatic hybrids to regenerate complete plants. Different methods of protoplast fusion are described by Bengochea and Dodds, (1986). Three important methods are generally used for protoplast fusion: (a) high Ca++ and high pH, (b) polyethylene glycol (PEG), and (c) electric field. Literature survey indicates that the second and third methods are most commonly used due to better results. The three methods are discussed below hybridization occurs when haploid cells generated in a previous meiosis fuse. The fusion of somatic diploid cells should generate a tetraploid fusion product provided that the nuclei fuse, too. If this is the case, it is spoken of a synkaryon. A fusion product where the nuclei stay separate is called a heterokaryon.
High Ca++ and High pH Induced Protoplast Fusion
Physical contact of two protoplasts is essential for their fusion. However, protoplasts do not fuse easily due to two main reasons: (i) they have a net negative charge on their membrane surfaces and force of repulsion works between protoplasts, and (ii) it is difficult to remove water from hydrophilic surfaces of protoplasts which also create a repulsive force between two protoplasts. It was observed that positively charged ions reduce the net negative charges of membranes reducing the repulsive force considerably. Keller and Melcher (1973) found calcium ions were suitable for such purposes and developed this method of protoplast fusion using high Ca++ ions in a high pH solution. Later the method was improved by Melcher and Labib (1974). The technique involves the following steps: Freshly isolated protoplasts of selected parents are mixed in a ratio of 1: with a final density of 2 5 protoplasts per ml. The protoplasts are collected as a pellet by centrifuging at 50 g for 3 to 5 minutes. The supernatant is removed and 2 ml of fusion mixture containing 50 mM CaCl 2 .2H 2 O, 50 mM Glycine-NaOH buffer and 400 mM Mannitol is added. pH of the mixture is adjusted to 10. Protoplasts are re- suspended in solution by gently shaking the centrifuge tubes protoplasts are collected by centrifuging at 50 g for 3 to 5 minutes. The centrifuge tubes with protoplasts are incubated in a water bath at 37°C for 10 to 30 minutes. The fusion mixture is replaced by washing medium (600 mM Mannitol and 50 mM CaCl 2 .2H 2 O) and protoplasts are left for 30 minutes protoplasts are washed twice with the washing medium protoplasts are re-suspended in culture medium. Protoplast fusion products can be observed under microscope. These protoplasts can be cultured on selection medium, which allows only somatic hybrids to grow.
PEG Induced Fusion
Polyethylene glycol (PEG) induced protoplast fusion was developed by Kao and Michayluk (1974). This technique is relatively more efficient than the previous one. Polyethylene glycol molecules have polarity like membrane phoshopholipid molecules and get attached with membrane proteins. When the attached PEG
between two protoplasts is removed it results in breakdown of membranes at the contact points causing protoplasts to fuse with subsequent rejoining of plasma membranes of the adjacent protoplasts. The following steps are involved for protoplast fusion through polyethylene glycol. Mix the freshly isolated protoplasts (while still in enzyme solution) of selected parents in a ratio of 1:1. Pass the suspension through a 62 μm pore size filter and collect the filtrate in a centrifuge tube. Seal the mouth of the tube with screw cap. Centrifuge the filtrate at 50 g for 60 minutes to sediment the protoplasts. Remove the supernatant with pasture pipette. Wash the protoplasts with 10 ml of solution I (solution I: 500 mM Glucose, 0 mM KH 2 PO 4 .H 2 O and 3 mM CaCl 2 .2H 2 O and pH 5). Remove and wash
the protoplasts in solution I and make a suspension with 4-5% v/v protoplasts per ml. Put a 2- 3 ml drop of silicon 200 fluid (100 cs) in a 60 x 15 mm sterile Petri dishes a 22x22 mm cover slip on the drop. Pipette 150 μl of protoplast suspension onto the cover slip with a pasture pipette. Allow about 5 minutes for the protoplasts to settle on the cover slip forming thin layer. Add drop-by-drop 450 μl of PEG solution (50% PEG-1540, 10 mM CaCl 2 .2H 2 O, 0 mM KH 2 PO 4 .H 2 O) to the protoplast suspension and observe the protoplast adhesion under inverted microscope. Inoculate the protoplast in PEG solution and keep at room temperature (24°C) for 10 to 20 minutes. Gently add 0 ml aliquots of solution II (50 mM Glycine, 50 mM CaCl 2 .2H 2 O, 300 mM Glucose, pH 9-10) at 10 minutes intervals. After another 10 minutes add 1 ml of protoplasts culture medium. Wash the protoplasts five times at five minutes intervals with 10 ml of the fresh protoplasts culture medium. At the end of each washing do not remove entire medium from the cover slip but leave behind a thin layer of the old medium adding fresh medium to it. If parent protoplasts are distinguishable visually, it may be possible to assess the frequency of heterokaryon formation at this stage. Culture the fused protoplasts together with the unfused protoplasts on the same cover slip in a thin layer of 500 μl of culture medium. Put additional 500-1000 μl medium in the front of droplets around the cover slip to maintain humidity inside the Petri plates.
Electric Field Induced Fusion
In this method, developed by U. Zimmerman, protoplasts are placed in an electric field and are exposed to high intensity electric pulse for a short duration (nano to micro second). This exposure to electric field reversibly increases permeability of cell membrane. Local electrical charge break down occurs in the plasma membranes resulting in fusion of adjacent protoplasts. The original properties of membrane are restored within micro second to minutes depending on the experimental conditions and membrane properties (Zimmerman and Scheurich, 1981; Zimmermann and Vienken, 1982). An instrument designed by U. Zimmerman is used for protoplast fusion and for genetic transformation through electroporation. The process of electric field induced protoplast fusion can be explained in the following five steps:
Dielectrophoresis Mutual dielectrophorasis Membrane contact Electric break down of membranes, and Protoplast Fusion
not move to either side due to equal electric force on both sides (Fig. 3A). However, under non uniform field (field strength on the two sides of particles is unequal) a net force acting upon the particle results in linear motion towards the region of highest field intensity (Fig. 3B). This is termed as dielectrophoresis. (ii)Mutual Dielectrophoresis : One protoplast approaching another polarized protoplast during the movement towards the region of highest field intensity, will encounter an enhancement of local field divergence and will tend to move towards that protoplast since the field strength is higher near that cell. As a result protoplasts in non uniform field form chain like aggregates (so called pearl chain) with point to point membrane contact (Fig. 3C). This effect is termed as mutual dielectrophoresis.
A. Uniform field developed equal force on both side of charged protoplasts B. Unequal electrical field developed net force towards higher field strengthC. Non uniform electric field thus forms pearl chain of protoplasts
Fig. 3. Movement of protoplast in non uniform electric field towards higher strength electric field and forms protoplast chain.
The attraction forces arising from dipole generation within protoplasts overcome both the electrostatic repulsive forces between membrane surfaces of neighboring protoplasts bearing net negative charges and repulsive hydration forces. Repulsive hydration force is assumed to be a consequence of work required to remove water from hydrophilic surface as the protoplasts approach one another. Dielectrophoresis and pearl chain formation usually have to be performed in virtually non conductive solution (conductivity less than 10-4 cm-1). (iii)Electric Break Down : Reversible electric breakdown in the zone of membrane contact is the primary process responsible for the initiation of fusion (Fig. 4). Phospholipids are arranged in a planer bi-layer into which peripheral or integral structural carrier proteins are embedded in mosaic like fashion. The lateral fluidity of phospholipids is very high, while the movement vertical to the membrane surface is severely limited. A flip-flop movement of lipids in this direction is, therefore, highly difficult. In an equivalent electrical circuit the membranes can be represented by a plate capacitor of specific capacitance (Cm) and resistance (Rm) connected in parallel (Fig. 4A). The aqueous external solution and polar heads of liquids represent the plate of capacitor, while membrane interior forms a dielectric with relative dielectric constant of 2 to 3. The resistance of aqueous external solution RE is in series with the membrane. The specific resistance of cell membrane is in the order of 102 to 10 4 Wcm-2, while that of specific membrane bilayer is three to four orders of
magnitude higher. The specific capacitances of artificial and biological membranes on the other hand are comparable of having values of 0 to 0 mFcm-2 and 1 mFcm- 2 , respectively.
Fig. 4. (A) Diagrammatic representation of Charge pulse technique, (B) Electric break down occurs at the poles of the cells and the zone of contact between two protoplasts.
It is known that capacitors can only be charged to a certain maximum voltage. Above this voltage level (which is dependent on separation of plates, common area between plates and dielectric material of plates) electrical (dielectric) breakdown is observed in the capacitor. Electrical breakdown is associated with an extreme increase in the electrical conductivity of the capacitor, which is usually irreversible i. the capacitor is destroyed. In the self-regenerating capacitors on the other hand the original resistance and capacitance is restored under certain experimental conditions. Biological membranes and artificial lipid bilayer behave in an analogous way to the electric breakdown of the self-regenerating capacitors described above. The reversible electrical breakdown of protoplast membranes leads to perturbation of membrane structure, which permits an exchange of materials between the protoplast and its environments. In figure 4B it is shown that the electric field effects the protoplast adhering to each other in the orientation to the field direction only. In this case, electrical breakdown occurs at the poles of the cells and zone of contact between two protoplasts.
Merits of Electrically Induced Protoplast Fusion
1 fusion process between any two protoplasts of different species in a mixed population can be followed under microscope. This would be of particular interest when producing hybridoma cells. 2. Fusion process is synchronous and extends over a short duration. So the hybrids do not lose the viability. 3. Viability can also be affected by fusogenic compounds, which are thus able to interact with total membrane surface in an uncontrolled manner. In this method there is no use of such fusogenic compounds. 4. Yield of somatic hybrids is very high. 5. During the fusion, loss of intercellular substances is generally very low.
Fig. 5. Flow chart showing selection of hybrids using selection medium on which only hybrid can grow.
Fig. 6. Flow Chart showing complementary selection of somatic hybrids on specific culture medium.
mutant was chlorophyll deficient that could not grow more than few inches. When protoplasts of these two mutants were fused the hybrid produced green colonies in culture and later on regenerated into full plants in low light intensity (800 lux).
Gleba et al. (1975) developed a similar complementation selection procedure that involves complementation between plastome and genome chlorophyll mutant and does not utilize light sensitivity as a selection marker. Cocking et al. (1977) used albino protoplasts of P. hybrida and green leaf mesophyll protoplasts of P. parodii (Fig. 6). These somatic hybrids included strict amphidiploids.
Group 3 - Mechanical Isolation : In this method heterokaryons are selected and isolated visually under microscope. This method is particularly useful for those interspecies fusions where the post zygotic incompatibility prevents sexual hybridization. In such cases of interspecies fusion chromosome elimination may result in failure to select the somatic hybrids, while other interspecies fusion may result in somatic hybrid protoplasts with no evidence of specific chromosome elimination. In these instances the stable conditions may fail to be maintained on organogenesis. Through this selection approach such barriers to whole somatic hybrid plant formation can be identified and perhaps overcome.
Automatic separator of somatic hybrids from mixed population of protoplasts after fusion process has been demonstrated. In this method parent protoplasts are stained with two different colour dyes and somatic hybrids exhibiting mixed colour are sorted.
Group 4 - Morphology of the plant after regeneration : In this method, the somatic hybrids are regenerated into whole plants. The morphological characters of the hybrid plants are compared with those of the two parental lines to establish the similarities. This method is time consuming and is possible where regeneration of plants and hybrids is well established.
(a)Datura (b) Petunia (c) Nicociana tabcum (d) Daccus carrota
9 successful regeneration of complete plants from somatic hybrid have been reported by (a)Takebe, I. et al (b) E. Cocking, (c) I. Vasil (d) J. Power 10. Who developed electric induced protoplast fusion technique? (a)P. S. Carlson (b) E. Cocking (c) I. K. Vasil (d) U. Zimmermann
Answers 1. (b) 2. (b) 3. (c) 4. (b) 5. (c) 6. (d) 7. (c) 8. (c) 9. (a) 10. (d)
Protoplast Culture and Somatic Hybridization
Course: Plant Physiology (BSB403)
University: Jamia Millia Islamia
Students also viewed
Related documents
- British Orientalism-History Assignment
- Politics, Political Science, Political Thought, Theory An Orientation
- A Brife Note On Nationalism's advantages and disadvantages
- Emergence and decline of arab nationalism
- Exploration of Rabindranath Tagore's The Home and the World as Socio-political Novel
- Muslim Mystics AND SUFI Silsilahs